PCR (polymerase chain reaction) yana ɗaya daga cikin fasahar haɓaka DNA na in-vitro, tare da tarihin sama da shekaru 30.

Kary Mullis na Cetus, Amurka ne ya jagoranci fasahar PCR a cikin 1983. Mullis ya nemi takardar shaidar PCR a 1985 kuma ya buga takarda ta farko ta PCR akan Kimiyya a cikin wannan shekarar.Mullis ya samu kyautar Nobel a fannin ilmin sinadarai a shekarar 1993 saboda aikinsa.

Ka'idodin asali na PCR

PCR na iya haɓaka gutsuttsuran DNA da aka yi niyya da fiye da sau miliyan ɗaya.Ƙa'idar tana ƙarƙashin ƙirar DNA polymerase, ta amfani da DNA strand DNA a matsayin samfuri da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun wuri don tsawo.Ana maimaita shi a cikin vitro ta matakai kamar denaturation, annealing, da tsawo.Tsarin DNA na 'ya mace wanda ya dace da samfurin mahaifa na DNA.

Daidaitaccen tsari na PCR ya kasu kashi uku:

1.Denaturation: Yi amfani da babban zafin jiki don raba nau'i biyu na DNA.Haɗin hydrogen tsakanin igiyoyin DNA guda biyu ya karye a babban zafin jiki (93-98 ℃).

2.Annealing: Bayan an raba DNA mai ɗaure biyu, rage zafin jiki ta yadda na'urar zata iya ɗaure ga DNA mai ɗaci ɗaya.

3.Extension: DNA polymerase yana farawa don haɗa nau'ikan maɗaukaki tare da igiyoyin DNA daga abubuwan da aka ɗaure lokacin da aka saukar da zafin jiki.Lokacin da aka gama tsawaita, ana kammala zagayowar, kuma adadin gutsuttsuran DNA ya ninka sau biyu

Maimaita waɗannan matakai guda uku sau 25-35, adadin guntuwar DNA zai ƙaru sosai.

Hazakar PCR ita ce, ana iya ƙera maɓalli daban-daban don nau'ikan kwayoyin halitta daban-daban, ta yadda za'a iya haɓaka gutsuttsuran kwayoyin halitta a cikin ɗan gajeren lokaci.

Ya zuwa yanzu, ana iya raba PCR zuwa kashi uku, wato PCR na yau da kullun, PCR mai kyalli da kuma PCR na dijital.

Farkon ƙarni na talakawa PCR

Yi amfani da kayan haɓakawa na PCR na yau da kullun don haɓaka asalin manufa, sannan amfani da agarose gel electrophoresis don gano samfurin, ƙididdiga masu inganci kawai za a iya yin.

Babban rashin amfani na ƙarni na farko na PCR:

1.Prone zuwa ba takamaiman haɓakawa da sakamako mai kyau na ƙarya.

2.A gano yana ɗaukar lokaci mai tsawo kuma aikin yana da wahala.

3.Only qualitative gwajin za a iya yi

Real-Time PCR na ƙarni na biyu

PCR na lokaci-lokaci, wanda kuma aka sani da qPCR, yana amfani da bincike mai haske wanda zai iya nuna ci gaban tsarin amsawa, kuma yana sa ido kan tarin samfuran da aka haɓaka ta hanyar tarin sigina mai kyalli, kuma yana yin hukunci da sakamakon ta hanyar lanƙwasa.Ana iya ƙididdige shi tare da taimakon ƙimar Cq da daidaitaccen lanƙwasa.

Saboda ana aiwatar da fasahar qPCR a cikin rufaffiyar tsarin, yuwuwar kamuwa da cuta ta ragu, kuma ana iya lura da siginar kyalli don gano ƙima, don haka ita ce mafi yawan amfani da ita a cikin aikin asibiti kuma ta zama babbar fasaha a cikin PCR.

Abubuwan da aka yi amfani da su a cikin PCR na gaske ana iya raba su zuwa: TaqMan bincike mai walƙiya, tayoyin kwayoyin halitta da rini mai kyalli.

1) TaqMan bincike mai kyalli:

A lokacin haɓakawa na PCR, ana ƙara takamaiman bincike mai kyalli yayin ƙara nau'i biyu na firamare.Binciken wani oligonucleotide ne, kuma duka ƙarshen suna da lakabi tare da ƙungiyar masu walƙiya mai ba da rahoto da ƙungiyar masu kyalli.

Lokacin da binciken ya kasance cikakke, siginar mai kyalli da ƙungiyar masu ba da rahoto ke fitarwa tana ɗaukar ƙungiyar masu kashewa;a lokacin haɓakawa na PCR, aikin 5'-3' exonuclease na Taq enzyme yana rarrafe kuma ya ƙasƙantar da binciken, yana sa ƙungiyar mai ba da rahoto ta ƙungiyar fluorescent da quencher Ƙungiyar fluorescent ta rabu, don haka tsarin kula da hasken wuta zai iya karɓar siginar hasken wuta, wato, duk lokacin da aka haɓaka DNA strand, an haɓaka siginar da aka yi da siginar da ke haifar da haɓakawa da haɓakawa gaba ɗaya. tare da samuwar samfurin PCR.

2) Rini mai kyalli na SYBR:

A cikin tsarin amsawar PCR, an ƙara yawan rini na SYBR mai kyalli.Bayan SYBR mai kyalli ba a haɗa shi ta musamman a cikin madauri biyu na DNA ba, yana fitar da siginar kyalli.Kwayoyin rini na SYBR wanda ba a haɗa shi cikin sarkar ba ba zai fitar da kowane sigina mai kyalli ba, don haka tabbatar da siginar kyalli Haɓaka samfuran PCR gaba ɗaya yana aiki tare tare da haɓaka samfuran PCR.SYBR kawai yana ɗaure ga DNA mai ɗaure biyu, don haka za a iya amfani da yanayin narkewa don sanin ko aikin PCR ya keɓanta.

3) Tashin hankali:

Wani bincike ne mai suna oligonucleotide mai tushe mai tushe wanda ya samar da tsarin gashin gashi na kusan sansanoni 8 a ƙarshen 5 da 3.Matsalolin acid nucleic a ƙarshen duka an haɗa su gaba ɗaya, yana haifar da ƙungiyar masu kyalli da ƙungiyar masu kashewa.Kusa, ba za a samar da haske mai haske ba.

Bayan da aka samar da samfurin PCR, yayin aikin cirewa, ana haɗe tsakiyar ɓangaren fitilar kwayar halitta tare da takamaiman jerin DNA, kuma an raba kwayar halitta mai walƙiya da kwayar halitta don samar da haske.

Babban rashin amfanin PCR na ƙarni na biyu:

Har yanzu ba a sami hankali ba, kuma gano ƙananan kwafi ba daidai ba ne.

Akwai tasirin ƙimar baya, kuma sakamakon yana da sauƙin shiga tsakani.

Lokacin da akwai masu hana PCR a cikin tsarin amsawa, sakamakon ganowa yana da sauƙin shiga tsakani.

PCR dijital na ƙarni na uku

PCR na dijital (DigitalPCR, dPCR, Dig-PCR) yana ƙididdige adadin kwafin jerin abubuwan da aka yi niyya ta hanyar gano ƙarshen ƙarshen, kuma yana iya yin daidaitaccen gano ƙididdige ƙididdigewa ba tare da amfani da sarrafawa na ciki da madaidaitan lanƙwasa ba.

PCR na dijital yana amfani da gano ƙarshen ƙarshen kuma baya dogara da ƙimar Ct (kofin sake zagayowar), don haka ƙimar PCR na dijital ba ta da tasiri ta haɓakar haɓakawa, kuma ana haɓaka haƙuri ga masu hana halayen PCR, tare da babban daidaito da haɓakawa.

Saboda halaye na babban hankali da daidaito mai girma, masu hana masu hanawa na PCR ba su da sauƙin shiga tsakani, kuma yana iya cimma cikakkiyar ƙididdigewa ba tare da daidaitattun samfuran ba, wanda ya zama wurin bincike da aikace-aikacen.

Dangane da nau'ikan rukunin naúrar amsawa, ana iya kasu kashi uku na manyan nau'ikan: microfluidic, coc da tsarin droplet.

1) Microfluidic dijital PCR, mdPCR:

Dangane da fasahar microfluidic, samfurin DNA ya rabu.Fasahar microfluidic na iya gane samfurin nano-haɓakawa ko samar da ƙananan ɗigon ruwa, amma ɗigon ruwa yana buƙatar hanyar talla ta musamman sannan a haɗa shi da tsarin amsawar PCR.mdPCR a hankali an karɓi ta ta wasu hanyoyin maye gurbin.

2) PCR na dijital na tushen Droplet, ddPCR:

Yi amfani da fasahar samar da ɗigon ruwa-cikin mai don sarrafa samfurin zuwa ɗigon ruwa, da kuma rarraba tsarin amsawa mai ɗauke da kwayoyin acid nucleic zuwa dubunnan ɗigon nanoscale, wanda kowannensu ba ya ƙunshi ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta da za a iya ganowa, ko kuma Ya ƙunshi ɗaya zuwa ƙwayoyin nucleic acid da yawa da za a gwada.

3) PCR dijital na tushen guntu, cdPCR:

Yi amfani da fasahar hanyar hanyar ruwa mai haɗaɗɗiya don zana microtubes da microcavities da yawa akan wafers silicon ko gilashin quartz, da sarrafa kwararar mafita ta bawul ɗin sarrafawa daban-daban, da raba ruwan samfurin zuwa nanometers na girman girman ɗaya a cikin rijiyoyin amsawa don amsawar PCR na dijital don cimma cikakkiyar ƙididdigewa.

Babban rashin amfani na PCR na ƙarni na uku:

Kayan aiki da reagents suna da tsada.

Abubuwan ingancin samfuri suna da girma.Idan samfurin samfurin ya zarce adadin microsystem, ba zai yuwu a ƙididdige shi ba, kuma idan ya yi ƙanƙanta, za a rage daidaiton ƙididdigewa.

Hakanan za'a iya haifar da tabbataccen ƙirƙira lokacin da babu takamaiman haɓakawa.